The protein distribution in the blood can indicate the overall state or condition of the human body. Therefore, determination of the proteins in blood samples can serve as a powerful diagnostic tool for early detection of disease states, such as cancer. Unfortunately, there is no analytical method available today to provide rapid high resolution profiles of proteins present in complex mixtures such as blood. The overall objective of this proposal is to develop a new method called electromobility focusing (EMF) for separating, concentrating, and identifying proteins in complex mixtures with unsurpassed resolution. The physical description of the device for EMF is a channel containing a buffer solution along which a high voltage is applied, similar to capillary electrophoresis, except that the electric field intensity along the column is not linear, but is a continuous gradient. Proteins can be separated and focused with exceptionally high resolution in the channel by applying a pressure-induced liquid flow counter to the electrophoretic migration direction of the proteins. The resulting forces acting on the proteins concentrate them into narrow bands at specific positions along the channel in order of their electrophoretic mobilities. The proteins can be mobilized in the separation channel by changing the magnitude of the pressure-induced counter flow or by reducing the applied voltage. The specific objectives of this work include the development of two different device formats for performing EMF: (a) semi-preparative and (b) microchip. EMF will be applied to the rapid quantitative detection of five known protein tumor markers in blood serum, including prostate specific antigen, carcinoembryonic antigen, carcinoma-associated antigen 1 25, alpha-fetoprotein, and humanchorionic gonadotropin. Proposed new tumor marker, thymidine kinase I, will also be studied.